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Laboratory Experiment

pubs.acs.org/jchemeduc

Rapid Determination of Enantiomeric Excess via NMR Spectroscopy:

A Research-Informed Experiment
John S. Fossey,*,† Eric V. Anslyn,‡ William D. G. Brittain,† Steven D. Bull,§ Brette M. Chapin,†,‡
Cécile S. Le Duff,† Tony D. James,§ Glenn Lees,† Stephanie Lim,‡ Jennifer A. C. Lloyd,†
Charles V. Manville,† Daniel T. Payne,† and Kimberley A. Roper†
†
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, West Midlands B15 2TT, U.K.
‡
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
§
Department of Chemistry, University of Bath, Bath, BA2 7AY, U.K.
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*
S Supporting Information

ABSTRACT: An undergraduate chemistry experiment that draws from primary research is

Downloaded via 88.19.176.204 on November 9, 2022 at 20:45:46 (UTC).

described. The experiment exploits chiral supramolecular assemblies for the determination of
enantiomeric excess by 1H NMR spectroscopy. This report describes the delivery of the
experiment to a cohort of students, and as a result of feedback from those involved, an
optimized protocol is presented. Particular care has been taken to facilitate ready adoption in
other institutions by providing comprehensive teaching support materials as well as technical
guidance for supporting the experiment.

KEYWORDS: Second-Year Undergraduate, Organic Chemistry, Analytical Chemistry, Laboratory Instruction,

Hands-On Learning/Manipulatives, Computer-Based Learning, Stereochemistry, Chirality/Optical Activity, Enantiomers,
NMR Spectroscopy

■ INTRODUCTION
Determining the enantiomeric excess (ee) of an asymmetric
transformation is an everyday task for synthetic chemists, and
being able to accurately measure the enantioenrichment of a
product is crucial to a wide range of chemistry,1 especially in
areas such as asymmetric catalysis.2
Being able to quickly and accurately measure the ee of a
reaction mixture has been a focus for development in industrial
and academic settings due to increased demand of quick ee
determination in high-throughput screening (HTS) method-
ologies.3 Established means of analysis (chiral HPLC) are not
ideal for these applications, where hundreds, if not thousands,
of asymmetric transformations need to be quantified daily,
requiring long method development and lengthy run times.
Proton nuclear magnetic resonance (NMR) spectroscopy is a
methodology that has been targeted for HTS because a proton
NMR spectrum can be obtained in under 5 min and thus can
reduce analysis times compared with established chiral
chromatography methods.4 Undergraduate students should be
aware of techniques that can form diastereomeric mixtures from
enantiomeric mixtures (e.g., Mosher’s acid derivitization) and
should develop an appreciation that these can be applied to ee
determination using NMR spectroscopy.5
However, undergraduate students do not often have
opportunities to gain practical experience in measuring ee.
There have been several articles and experiments published
within this journal which look at chirality and its quantitation,
© 2016 American Chemical Society and
Division of Chemical Education, Inc.
however none use 1H NMR spectroscopy as a methodology in
which to quantify ee.6 We wished to tackle this through
research-informed teaching, applying cutting edge ee determi-
nation methodology to reinforce multidisciplinary aspects of
undergraduate learning, such as supramolecular, organic, and
analytical chemistry. Developing an undergraduate practical
procedure based upon chiral HPLC could be both time-
consuming and expensive, especially in institutions that do not
already possess chiral HPLC capacity. Therefore, we have
developed an undergraduate protocol for determination of ee
based on 1 H NMR spectroscopy and supramolecular
assemblies, using commercially available materials, and
demonstrating the impact of real life research in the
undergraduate laboratory.7 The utilization of NMR spectros-
copy can be viewed as a “green” alternative to HPLC due to the
reduction in solvent used per sample (0.6 mL for NMR
spectroscopy vs ca. 60 mL for HPLC), and the acquisition time
is reduced from up to 60 min for HPLC to 5 min for a 1H
NMR experiment. Any institution which possesses a 200 MHz
or greater NMR spectrometer will be able to integrate this
experiment into its undergraduate course.
Received: April 29, 2016
Revised: August 26, 2016
Published: September 30, 2016
79 DOI: 10.1021/acs.jchemed.6b00355
J. Chem. Educ. 2017, 94, 79−84
Journal of Chemical Education
This experiment takes advantage of a boronic acid based
assembly, which was reported by Bull, James, and co-workers
(Scheme 1).8 The Bull−James assembly utilizes the ability of a
Scheme 1. Bull−James Assembly Used for the
Determination of EE of Primary Amines
boronic acid to bind a diol, as well as an ortho-formyl group
which can condense with a primary amine to form an imine
operating cooperatively to form a three-component assembly.
Using a stereodefined diol (e.g., (R)-1,1′-bi-2-naphthol
(BINOL) (2)) with 2-formylphenylboronic acid (2-FPBA)
(1) (Scheme 1), the ee of a primary amine can be inferred
through measurement of the ratio of diastereoisomers formed
as observed through NMR spectroscopy. Using this type of
assembly introduces undergraduate students to the concepts of
enantiomeric excess, chiral shift reagents, and chiral derivatizing
agents while building on their knowledge of stereoisomerism
(particularly the difference between enantiomers and diaster-
eomers and their respective spectroscopic properties).
Upon addition of a chiral primary amine to 2-FPBA (1), an
imine is formed, reinforcing undergraduate understanding of
carbonyl chemistry, which is a mainstay of organic under-
graduate lectures. Subsequent binding of the chiral diol (in this
case commercially available (R)-BINOL (2)) to the boronic
acid forms a boronate ester, causing the formation of a three-
component assembly, potentially as a mixture of diaster-
eoisomers proportional to the enantiomeric ratio of the original
chiral primary amine (Scheme 2).9 The integration of the peaks
in the 1H NMR spectrum corresponding to the two
diastereoisomers formed thus infers the ee of the chiral primary
amine initially used. This assembly has been shown to be
applicable to the determination of ee of a wide variety of
different primary amines. For this experiment commercially
available α-methylbenzylamine (4) was chosen, however we
anticipate that this experiment could be modified easily to
accommodate a range of primary amines.
The learning outcomes and pedagogic goals of this
experiment are to offer practical experience with ee
determination; this experiment also introduces undergraduate
students to NMR sample preparation and processing software.
Scheme 2. Boronic Acid Based Three-Component Assembly for the EE Determination of α-Methylbenzylamine
80
Laboratory Experiment
Students will become comfortable with manual integration of
1
H NMR spectra, which is taught in spectroscopy courses.
Giving students experience in the processing of NMR spectra is
a good introduction into how NMR data are analyzed in
research laboratories.
This experiment has been trialed at the University of
Birmingham (U.K.) with a cohort of 113 second-year
undergraduate chemists during their second semester. The
experiment was carried out under the following conditions:
students carried out the laboratory section within a fume hood
lab at the University of Birmingham (U.K.) while the data
analysis was carried out using a computer cluster at the same
institution. The following software was used: Microsoft Excel or
Sigmaplot (graphing software), MNova or TopSpin (NMR
processing, TopSpin is available for free for academic use),
Microsoft Word (word processing), and Canvas (online
learning platform).10 This protocol includes developments
and improvements that were made as a result of observations
and feedback from the students.
■
EXPERIMENTAL OVERVIEW
Laboratory Practical
We suggest that students work in groups of five. The procedure
detailed here begins with preparation of a series of known ee
mixtures of α-methylbenzylamine (4) and a “host” solution of
2-FPBA (1) and BINOL (2). We recommend that each group
make a stock solution of host to which each amine solution is
then added. In order to construct a calibration curve, students
are given a table of seven ee values (ranging from −75% ee to
+75% ee)11 and corresponding volumes of (R)- and (S)-α-
methylbenzylamine (4) in order to make solutions of the
tabulated ee values (see the Laboratory Manual). Each person
in the group of five will prepare one of the seven suggested
solutions, so that the calibration curve will contain five points.
We ask the students to think critically about which ee mixtures
are the most sensible choices to contribute to their calibration
curve. Students use automated pipettes to transfer α-
methylbenzylamine (4) into 10 mL volumetric flasks and
then dilute using chloroform-d. The host solution is obtained
by weighing out the solid 2-FPBA (1) and (R)-BINOL (2)
followed by dissolution in chloroform-d in a 10 mL volumetric
flask. The solutions of amine are prepared at 60 mM, while the
host solution is 50 mM in both 2-FPBA (1) and (R)-BINOL
(2). To all of these solutions are added 4 Å molecular sieves,
and the solutions are allowed to dry for 10 min. The students
are also provided with a selection of seven 60 mM amine
solutions of unknown ee prepared before the laboratory period
by the instructor or a laboratory technician. Each student in the
group of five chooses one of these samples to combine with the
host solution and determine its ee. To make the samples ready
for NMR spectroscopic analysis, 0.3 mL of the known or
unknown amine solution is combined with 0.3 mL of the host
DOI: 10.1021/acs.jchemed.6b00355
J. Chem. Educ. 2017, 94, 79−84
Journal of Chemical Education
solution in an NMR tube. As designated, each student is
responsible for making two NMR samples (one with an amine
solution of known ee, and one with unknown ee). The samples
are then analyzed by 1H NMR spectroscopy (collection
parameters are described in the Laboratory Manual). It was
found that the practical part of the experiment described above
could be carried out comfortably within 2 or 3 h.
Data Analysis
The second part of this experimental procedure takes place at a
computer terminal, ideally in a teaching ready computer room.
The NMR data recorded from the samples prepared during the
laboratory session are returned to the students in electronic
format and then processed using NMR processing software.
The students are also provided with printed NMR spectra of
each of the three components as pure compounds in
chloroform-d. They are instructed to identify and compare
several key resonances: the aldehyde peak (OCH) in 2-
FPBA (1) and the imine peaks (NCH) of the diaster-
eoisomers (R,R)-5 and (R,S)-5 of the assembly, the benzylic
protons in the free amine 4 and in the assembly (R,R)-5 and
(R,S)-5, and the alcohol peak (OH) in (R)-BINOL (2).
Students then use the NMR processing software to analyze the
relative integrations of peaks in the imine and benzylic regions
of their group’s spectra to determine the ratio of
diastereoisomers (R,R)-5 and (R,S)-5 and infer the ee of the
starting α-methylbenzylamine 4 mixture in their unknown. The
students will need to construct two calibration curves (one
using the imine protons and one using the benzylic protons)
using the spectra of the known ee samples they made as a team.
Plotting the observed ee vs the true ee should garner a straight
line, and an equation of best fit can be calculated. This equation
can then be used to determine the ee of the five unknown
amine samples the students have made. The students should
plot their own calibration curve using the data from the five
students in the team. In the event of some error in sample
preparation, instructors may have to substitute data so students
can carry out the analysis. Calibration curves for both possible
sets of protons (imine and benzylic) should be plotted.
Students may find one calibration curve gives a better straight-
line correlation and therefore may choose to select one of the
two curves to determine their ee values. Students should
comment on the accuracy and reproducibility of the data
obtained or discarded and then make an informed decision on
which data set they believe to be a more accurate measure of
the true ee. The proforma to be filled out by the students asks
them to justify the decisions they have made throughout the
data analysis and also discuss the chemistry involved in this
process.
■
HAZARDS
All hazards are outlined in the laboratory manual,12 however it
is best if the students are reminded of all the below safety issues
before beginning the experiment. Safety glasses, nitrile gloves,
and a laboratory coat should be worn at all times in the
laboratory, and care should be taken when handling the
chemicals. Both (R)- and (S)-α-methylbenzylamine (4) can
cause severe skin burns and eye damage, and the (S)-
enantiomer is also toxic in contact with skin. 2-FPBA (1) is a
skin, eye, and respiratory irritant. (R)-BINOL (2) is toxic if
swallowed, however any risk of ingestion carrying out this
procedure is minimal. During the experiment students will be
handling significant volumes of deuterated chloroform, which is
81
Laboratory Experiment
harmful if swallowed and also suspected of causing cancer. For
this reason, students should work in a fume hood when
handling chloroform. Acetone is also used to wash glassware;
students therefore should be aware that it is flammable and can
cause eye irritation in addition to feelings of drowsiness or
dizziness. Diastereoisomers (R,R)-5 and (R,S)-5 also need to be
considered for their hazards: as material safety data sheets
(MSDS) are not available for these products, students should
assume that they are toxic, irritating, and corrosive and thus
handle accordingly. However, these products are made in a
sealed NMR tube, and thus exposure risk should be minimal.
When carrying out any laboratory work it is vital that local
health and safety procedures are observed. Therefore, before
carrying out this experiment in another undergraduate
laboratory the process should be assessed to ensure that it
adheres to local health and safety requirements.
■
RESULTS AND DISCUSSION
This experiment has been successfully run with a cohort of 113
students at the University of Birmingham (U.K.). A few minor
alterations to the earlier versions of this experiment were made
during testing. It was found that students often spilled a small
amount of their NMR solutions down the outsides of the NMR
tubes while making up their NMR samples, making the tubes
unpleasant to handle for the NMR staff running the samples.
However, more importantly, soiled tubes are a problem for
NMR spectroscopy as dirt will accumulate in the probe and on
the spinners over months of intensive use, therefore decreasing
the quality of the data obtained. Therefore, instructors should
ensure that students wipe the outside of their NMR tubes with
a tissue soaked with a small amount of acetone to remove any
residue. Direct measurement using graduated pipettes into
NMR tubes should be discouraged due to the propensity for
NMR tube breakage. Therefore, it is recommended that
students make up their solutions in glass vials using a graduated
glass pipet, before then transferring an aliquot of this solution
into the NMR tube using a Pasteur pipette in the usual fashion.
The most critical aspect for obtaining good quality data is the
removal of adventitious water from the NMR samples. It was
found that the addition of activated 4 Å molecular sieves to all
of the solutions used in this experiment was key to obtaining
good quality NMR spectra. This enabled accurate determi-
nation of diastereomeric ratios, which gave inferred ee values
that were accurate to within ±10% of the true ee values in the
hands of undergraduate students. Any residual water can
hydrolyze the formed imine and can potentially compete with
the (R)-BINOL (2) to bind with the boron atom.
Use of the correct ratio of amine solution to host solution is
important to obtain accurate diastereomeric ratios by NMR
spectroscopic analysis. More specifically, it is important that the
amine is in excess and that kinetic resolution based on the
preferential formation of one diastereoisomer is not observed.8e
If the host solution is in excess, residual BINOL (2) can
produce a very broad peak (corresponding to the phenolic
protons) in the same region of the 1H NMR spectrum as the
benzylic protons of the diastereoisomers created upon assembly
formation. Therefore, the presence of excess BINOL (2) leads
to a benzylic region which cannot be used to accurately
determine the true ee value of the starting amine due to overlap
between free BINOL (OH) and benzylic resonances. Some
students observed this phenomenon, possibly due to inaccurate
preparation of host and/or amine solutions. In cases where this
occurs (this was a rare occurrence; around 1 group in 5 may
DOI: 10.1021/acs.jchemed.6b00355
J. Chem. Educ. 2017, 94, 79−84
Journal of Chemical Education
have an anomalous point), after the student has plotted the
calibration curve and observed that there are anomalous points
(which obviously do not fit a linear regression), if there is time
to repeat the experiment this is of course the best method to
remedy inconsistent data. However, where there are lab space
and time restraints in order to complete the experiment in a
timely manner and for students to get accurate ee
determinations, instructors may guide groups to ignore points
in the calibration, or offer replacement data as they deem
appropriate (students are never encouraged to ignore data).
Such observations are also incorporated into the assessment, as
students are asked to critically evaluate the quality of their data
and make judgements as to whether it is ever appropriate to
omit data points. This critical analysis is pivotal within a
research environment, so developing these skills during
undergraduate learning is important to their future success in
a research setting.
During the data analysis session, it was noted that a thorough
introduction into the use of NMR processing software was
required. The majority of students had no experience with
NMR data processing and struggled to use the programs
effectively without precise instructions. The Student Laboratory
Manual includes a short introduction to MNova 10 and
TopSpin, and video files demonstrating the basic functions of
MNova and TopSpin are provided; however, any program
which can open and integrate NMR data can be used.
■ REFLECTIONS
This experiment has been carried out by a cohort of 113
second-year undergraduate students (groups of 20 students
took part in the experiment over a series of 6 weeks) at the
University of Birmingham (U.K.). This exercise was part of a
general undergraduate laboratory program carried out during
the second semester, however this procedure could potentially
be used as part of an organic, physical organic, or analytical
chemistry course. During the testing of this experiment we
learned several ways in which our initial protocol could be
improved, and these improvements have been implemented
into the described experimental procedures.
Students were required to fill in a proforma for this
experiment, and several observations were made from the
marking of these assessments. First, the construction of the
calibration curves was achieved with variable success. A key
training point was the construction of the calibration graphs.
Despite difficulties in the construction of the graphs students
were successful in applying these graphs to determine the ee of
unknown samples. Students found it difficult to deal with
hypothetical situations, such as the reasons for potential error
being introduced into this experiment, with many students
constructing answers relating to human-based errors, or specific
examples that they encountered. The essay style questions were
designed to challenge the critical thinking of the students and
to encourage them to assess and question the validity of the
data they obtained; some students found it challenging to
answer these questions. Some were reluctant to comment on
the quality of their data, perhaps feeling that critical self-
assessment would lead to a loss of marks. However, the mark
scheme is specifically designed to encourage the critical
evaluation of data, regardless of results obtained, and students
may need to be reassured of this during the experiment.
The student experience from this experiment appeared
mainly positive. Each student was given a feedback ques-
tionnaire about what they liked and disliked about the protocol.
82
Laboratory Experiment
In the majority they found the laboratory instructions to be
clear and easy to follow. They did note that, due to this being
the first time that they experienced using NMR processing
software, they found it confusing in places. Therefore, in this
revised version we have produced written and video
instructions for software use.
Overall, this protocol demonstrated a practical introduction
into the use of 1H NMR spectroscopy as a technique for
determining the ee of a primary amine, via diastereoisomer
formation, including an introduction to the use of NMR
processing software. In addition to this, the experiment reminds
students of the relationships between diastereoisomers and
enantiomers and reinforces knowledge of carbonyl condensa-
tion chemistry with amines. It also introduces them to the
reversible, covalent, supramolecular interactions between
boronic acids and diols13 and aims to encourage students to
critically analyze their decisions and results, both in the
laboratory and during data analysis.
■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available on the ACS
Publications website at DOI: 10.1021/acs.jchemed.6b00355.
Student laboratory manual (PDF, DOCX)
Technician notes (PDF, DOCX)
Demonstrator notes (PDF, DOCX)
Proforma assessment (PDF, DOCX)
Marking guide, for the proforma (PDF, DOCX)
NMR analysis software videos (ZIP)
Reference NMR data (ZIP)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: j.s.fossey@bham.ac.uk.
Author Contributions
All authors contributed to the preparation of the manuscript.
J.S.F. is the first and corresponding author; all other authors are
listed alphabetically. J.S.F. designed the undergraduate experi-
ment and conceived this manuscript. E.V.A., S.D.B., and T.D.J.
provided academic input toward the experiment and its original
research-inspired concepts. B.M.C., D.T.P. and W.D.G.B. wrote
the body of text and contributed toward the Supporting
Information. B.M.C. and J.A.C.L. investigated the experimental
methodology. W.D.G.B. provided laboratory instruction and
undergraduate support to the first cohort of students. D.T.P.
marked and provided feedback on the assessment of the first
cohort of students. C.S.L.D. and C.V.M. designed the NMR
analysis and data acquisition procedures. K.A.R. produced the
NMR software videos. G.L. contributed toward the production
of the Supporting Information. S.L. ran a preliminary version of
the experiment and provided feedback.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors would like to thank Katie Topham and Rachel
Sturman (University of Birmingham Undergraduate Laboratory
Technicians), Ian Shannon (University of Birmingham Director
of Undergraduate Laboratories), John Snaith (University of
Birmingham Head of Education), and Wenlei Zhai for support
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J. Chem. Educ. 2017, 94, 79−84
Journal of Chemical Education
in the running of this experiment. J.S.F. would like to thank the
Royal Society for an Industrial Fellowship and the EPSRC for
funding (EP/J003220/1). J.S.F., E.V.A., W.D.G.B., and B.M.C.
are particularly grateful for a Royal Society International
Exchange Grant that facilitated much of this investigation.
E.V.A. also thanks the Welch Regents Chair for support (F-
0046). J.S.F., D.T.P., and W.D.G.B. would like to thank the
University of Birmingham for support. D.T.P. and W.D.G.B.
would like to thank the Royal Society of Chemistry and the
Society for Chemical Industry for travel grants. The Catalysis
and Sensing for our Environment (CASE) group is thanked for
providing essential networking opportunities.14 The second-
year undergraduate class of chemistry students (2015−2016) at
the University of Birmingham (UK) are thanked for under-
taking this experiment with their usual academic enthusiasm
and are awesome.
■
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enantiomeric purity of chiral primary amines. Nat. Protoc. 2008, 3,
210−214. (c) Kelly, A. M.; Bull, S. D.; James, T. D. Simple chiral
derivatisation protocols for NMR analysis of the enantiopurity of 1,2-
diphenylethane-1,2-diamine and N-Boc-cyclohexane-1,2-diamine. Tet-
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1971−1974. (e) Pérez-Fuertes, Y.; Kelly, A. M.; Johnson, A. L.;
Arimori, S.; Bull, S. D.; James, T. D. Simple Protocol for NMR
Analysis of the Enantiomeric Purity of Primary Amines. Org. Lett.
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analysis of the enantiopurity of O-silyl-1,2-amino alcohols. Tetrahedron
Lett. 2009, 50, 876−879. (g) Tickell, D. A.; Mahon, M. F.; Bull, S. D.;
James, T. D. A Simple Protocol for NMR Analysis of the Enantiomeric
Purity of Chiral Hydroxylamines. Org. Lett. 2013, 15, 860−863.
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Derivatives with Molecular Self-Assemblies. Angew. Chem., Int. Ed.
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Fossey, J. S.; Bull, S. D. A chiral ligand mediated aza-conjugate
addition strategy for the enantioselective synthesis of β-amino esters
that contain hydrogenolytically sensitive functionality. Tetrahedron
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chiral amine recognition and rapid ee determination. Chem. Sci. 2012,
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DOI: 10.1021/acs.jchemed.6b00355
J. Chem. Educ. 2017, 94, 79−84
Journal of Chemical Education Laboratory Experiment

(10) For more information on Canvas see https://www.canvaslms.

com/k-12/ (accessed Aug 2016).
(11) −Ee and +ee refer to opposite enantiomer in excess. −100% ee
is defined as a solution of pure (S)-MBA, and + 100% ee is defined as a
solution of pure (R)-MBA.
(12) Hazards noted here are taken from material safety data sheets
produced by Sigma Aldrich for compounds for sale in the United
Kingdom. These hazards may be classed differently in other countries.
(13) (a) Sun, X.; James, T. D. Glucose Sensing in Supramolecular
Chemistry. Chem. Rev. 2015, 115, 8001−8037. (b) Zhai, W.; Sun, X.;
James, T. D.; Fossey, J. S. Boronic Acid-Based Carbohydrate Sensing.
Chem. - Asian J. 2015, 10, 1836−1848.
(14) (a) Payne, D. T.; Fossey, J. S.; Elmes, R. B. P. Catalysis and
Sensing for our Environment (CASE2015) and the Supramolecular
Chemistry Ireland Meeting (SCI 2015): Dublin and Maynooth,
Ireland. 8th−11th July. Supramol. Chem. 2016, DOI: 10.1080/
10610278.2016.1150595. (b) Fossey, J. S.; Brittain, W. D. G. The
CASE 2014 symposium: Catalysis and sensing for our environment,
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84 DOI: 10.1021/acs.jchemed.6b00355
J. Chem. Educ. 2017, 94, 79−84